Insecticide based on a virus from the baculovirus group, and use thereof for the destruction of phthorimaea operculella

ABSTRACT

The invention is a method for destroying Phthorimaea operculella by causing the Phthorimaea operculella to ingest the baculorirus of the nuclear polyhedrose of the noctuid Mamestra brassicae.

The present invention relates to an insecticide agent active againstPhthorimaea operculella, an insecticide composition containing thisagent, a method of preparing the latter and a method of destroyingPhthorimaea operculella.

Phthorimaea operculella is a parasite of cultivated solanaceous plantssuch as potatoes, tobacco, tomatoes and aubergines. Thus, it is commonlycalled potato mite or tobacco leaf miner. The caterpillars of thisparasite feed on the plants on which they develop and man has alwaysfought against these harmful insects. For this, use has in particularbeen made of chemical insecticides whose toxicity with regards tovertebrae in general and Man in particular leads however to having totake strict precautions in use.

Thus, the aim of the present invention is to provide an insecticideinoffensive for vertebrae and plants and which may be termed biological.This invention is based on the surprising discovery that the naturalvirus (baculovirus of nuclear polyhedrose) of the noctuid Mamestrabrassicae (cabbage parasite) when it is ingested by the Phthorimaeaoperculella caterpillars causes considerable mortality among thesecaterpillars.

Consequently, the first object of the present invention resides in aninsecticide agent active against Phthorimaea operculella, which ischaracterized in that it is formed by the baculovius of the nuclearpolyhedrose of the noctuid Mamestra brassicae.

Since the baculoviruses generally have a very specific viral activity,it is quite unexpected that the particular baculovirus used in thepresent invention has an activity with respect to a parasite, thePhthorimaea operculella caterpillar, so different from the caterpillarof the noctuid Mamestra brassicae. In fact, Phthorimaea operculella andMamestra brassicae belong to different families, the first belonging tothe family of the noctuidae and the second to the family of theGelechiidae. Moreover, the baculovirus of Mamestra brassicae and thenatural virus of Phthorimaea operculella have different taxonomicpositions since the first enters in to the sub-group of the viruses ofnuclear polyhedroses whereas the second enters into the sub-group of theviruses of granulosis. It is also important to note that, since thegeographical areas in which Mamestra brassicae and Phthorimaeaoperculella are present are very different, there cannot be naturalmortality of Phthorimaea operculella due to the presence of the Mamestrabrassicae virus. A second object of the present invention resides in aninsecticide composition active against Phthorimaea operculella, thiscomposition being characterized in that it comprises, as activesubstance, the above defined insecticide in an appropriate vehicle.

This insecticide composition may be in different forms. Thus, it may forexample be in the form of a suspension of said insecticide agent in aliquid vehicle containing possibly a dispersant, this liquid vehiclebeing chosen preferably from the group comprising water, vegetable oils(particularly cotton oil) and mixtures thereof. It may in a variant bein the form of a powder comprising said insecticide agent and a powderyvehicle such as talc. Of course, this composition may if desired alsocontain other additives, e.g. additives capable of improving adherence.

The present invention further has as object a method of preparing theabove defined insecticide agent, this method being characterized in thatit comprises the steps which consist:

(a) in contaminating Mamestra brassicae caterpillars in the last but onelarval stage with the baculovirus of the nuclear polyhedrose of thenoctuid Mamestra brassicae, and

(b) extracting from the caterpillars which have died following suchcontamination the baculoviruses which have increased therein.

Advantageously, step (a) of this method consists in causing saidcaterpillars to ingest a nutritional medium contaminated with saidbaculoviruse, this medium comprising preferably the followingnutritional elements: cabbage powder, beer yeast, crushed maize, crushedwheat germ, casein hydrolysate, mineral salts.

As for step (b) of this method, it preferably comprises the crushing ofthe dead caterpillars and the filtration of the slurry resulting fromsuch crushing, on a filter having a mesh opening capable of retainingparticles of a dimension greater than 0.3 μ and particularly thepolyhedres of the baculovirus which have a dimension of 0.5 to 2.5 μ,such filtration being followed by elimination of the liquid phase of thefiltrate. Such elimination may in particular be carried out by acetonicprecipitation int he presence of lactose, lyophilisation or spraydrying.

The present invention finally has as object a method of destroyingPhthorimaea operculella infesting the foliage, the tubercles, the fruitsand/or the flower buds of cultivated solanaceous plants, this methodbeing characterized in that it consists in applying the above definedinsecticide agent or insecticide composition on said foliage, tubercles,fruits and/or flower buds. When the insecticide composition is used inthe form of a suspension, it is preferably applied on the foliage,fruits and/or flower buds at the rate of 1.10¹³ to 5.10¹³polyhedres/hectare of foliage, fruits and/or flower buds and when it isused in the form of a powder, the latter is preferably deposited bypowdering on the tubercles at the rate of 1.10⁹ to 10.10⁹ polyhedres/gof tubercles.

The following non limitative examples are given by way of illustrationof the present invention.

EXAMPLE 1

Production of the baculovirus of the nuclear polyhedrose of the noctuidMamestra brassicae.

Mamestra brassicae caterpillars are raised on an artificial nutritionalmedium (M) described in example 2, until the last but on larval stage.They are then infected by ingesting an artificial nutritional medium(M') described in example 3, contaminated by the baculovirus of thenuclear polyhedrose of the noctuid Mamestra brassicae. The contaminatedcaterpillars die within 9 to 10 days. The bodies are then crushed andfiltered for example on a glass fiber filter having a mesh size capableof retaining all particles of a dimension greater than 0.3 μ. On thefiltrate, an acetonic precipitation is carried out by adding theretofour volumes of acetone for one volume of filtrate and lactose at therate of 6% by weight of the filtrate and by subjecting the resultantmixture to energetic agitation for homogenizing it. As a variant, thefiltrate may be lyophilized or subjected to spray drying. The powderthus obtained contains the desired baculoviruses plus cellular waste.The activity of the preparations obtained as described above isdetermined by a series of counts using a hematimetric cell which makesit possible to count an average number of polyhedres, then by biologicaltests measuring the insecticide activity on caterpillars.

EXAMPLE 2

Composition of the nutritional medium (M) mentioned in example 1.

For 1 kg of this nutritional medium, the following are used:

    ______________________________________                                        Binders (A)                                                                   agar--agar                 16     g                                           distilled water            780    ml                                          Nutritional elements (B)                                                      cabbage powder             64     g                                           beer yeast                 34     g                                           crushed maize              64     g                                           crushed wheat germ         32     g                                           casein hydrolysate         5      g                                           saline mixture             3.5    g                                           Antimicrobial agents (B')                                                     benzoic acid               2.5    g                                           Nipagine (C) = Methyl parahydroxybenzoate                                                                1.1    g                                           Formaldehyde (D)           0.5    ml                                          Vitamins (E)                                                                  vitamin mixture            3      g                                           ascorbic acid              4.5    g                                           ______________________________________                                    

the agar-agar is dissolved in half the amount of water indicated, at120° C. for 20 minutes in autoclave.

Meanwhile, the powders B and B' are mixed with the remaining water; themethyl parahydroxybenzoate (C) is dissolved in 5 ml of 90° alcohol.

When the gelose is dissolved it is incorporated in the nutritionalelements (B) and (B') using a household type mixer. The temperature ofthe mixture then drops rapidly to 55° C., at which temperature theantimicrobial agents (C) and (D) and the vitamins (E) can be included.

The mixture, still hot, is distributed in the different raising devicesand rapidly hardens when cooling.

It may be kept in closed boxes for 8 to 10 days at 5° C.

EXAMPLE 3

Composition of the nutritional medium (M') mentioned in example 1.

This medium is also used for the biological tritration tests. For akilogram of medium the following are used: agar-agar 12 g, water 800 ml,cabbage powder 64 g, beer yeast 34 g, maize 64 g, wheat germ 32 g,benzoic acid 2.5 g, nipagine 1.1 g, ascorbic acid 4.5 g.

EXAMPLE 4

Treatment against potato mite.

The strain of the ravager used for the experiments comes from the DarBouazza region of Casablanca (Morocco). This strain is raised on potatotubercles, at a temperature of 25° C.

A test was carried out on potato leaves. Phthorimaea operculella mothsfrom Morocco are placed for laying in a cage containing branches withleaves and after 48 hours the eggs are counted. Then, on the leaves issprayed, in a treatment tower a suspension in water of the virusprepared in accordance with example 1 and at two different doses of thevirus, namely at the dose of 50 polyhedres/mm² of foliage (dose 1) andat the dose of 250 polyhedres/mm² of foliage (dose 2). The lethalactivity of this suspension is evaluated by counting the number of mothsformed from the eggs. The results obtained are the following:

    ______________________________________                                        untreated control 65 eggs lead to 54 moths:                                                     about 17% mortality                                         baculovirus dose 1                                                                              51 eggs lead to 37 moths:                                                     about 28% mortality                                         baculovirus dose 2                                                                              51 eggs lead to 17 moths:                                                     about 60% mortality                                         ______________________________________                                    

With this method of treatment, action of the virus related to the dosecan be observed.

Furthermore, several tests wee carried out on potato tubercles usingrespectively a powder formed of 98% by weight of talc and 2% of thebaculovirus prepared in accordance with example 1 and having a viralactivity of 72.10⁹ polyhedres/g and a powder formed of 90% by weight oftalc and 10% by weight of the same baculovirus. The amount of powderused represented 1% of the weight of the tubercles. The tubercles weretreated by coating them with the above powders and the lethal activityof these powders was determined by counting the number of moths formedfrom the eggs laid in 24 hours by 30 Phthorimaea operculella moths. Theresults obtained are as follows:

    ______________________________________                                        untreated control                                                                             100% of the eggs laid led to                                                  the formation of a moth                                       powder dosed at 2%                                                                            26% only of the eggs laid led                                                 to the formation of a moth                                                    (mortality rate 74%)                                          powder dosed at 10%                                                                           9% only of the eggs laid led                                                  to the formation of a moth                                                    (mortality rate 91%).                                         ______________________________________                                    

The above tests were repeated four times using an untreated control andthe powder dosed at 2% and the following percentages of moths wereobserved: control: 100%; powder dosed at 2%: 5%, 12%, 8% and 23%.

The above results undeniably demonstrate the powerful insecticideactivity of the compositions tested.

We claim:
 1. A method for destroying Phthorimaea operculella comprisingthe step of causing the Phthorimaea operculella to ingest thebaculovirus of the nuclear polyhedrose of the noctuid Mamestrabrassicae.
 2. The method of claim 1, wherein the baculovirus of thenuclear polyhedrose of the noctuid Mamestra brassicae is used in theform of a suspension in an appropriate liquid vehicle.
 3. The method ofclaim 1, wherein the baculovirus of the nuclear polyhedrose of thenoctuid Mamestra brassicae is used in the form of a mixture with apowdery vehicle.
 4. A method for the destruction of Phthorimaeaoperculella infesting the foliage, the tubercles, the fruits or theflower buds of cultivated solanaceous plants, which comprises applyingthe baculovirus of the nuclear polyhedrose of the noctuid Mamestrabrassicae on said foliage, tubercles, fruits or flower buds.
 5. Themethod of claim 4, wherein the baculovirus of the nuclear polyhedrose ofthe noctuid Mamestra brassicae is used in the form of a suspension in anappropriate liquid vehicle and is applied to the foliage, fruits orflower buds of said plants.
 6. The method of claim 5, wherein saidliquid vehicle is selected from the group consisting of water, vegetableoils and mixtures thereof.
 7. The method of claim 5 or 6, wherein saidsuspension is applied at the rate of 1.10¹³ to 5.10³ polyhedres/hectareof said foliage, fruits or flower buds.
 8. The method of claim 4,wherein the baculovirus of the nuclear polyhedrose of the noctuidMamestra brassicae is used in the form of a mixture with a powderyvehicle and is applied to the tubercles of said plants.
 9. The method ofclaim 8, which comprises powdering the tubercles with said mixture atthe rate of 1.10⁹ to 10.10⁹ polyhedres/g of tubercles.
 10. Aninsecticide composition for destroying Phthorimaea operculellacomprising as the active ingredient the baculovirus of the nuclearpolyhedrose of the noctuid Mamestra brassicae.